Our laboratory is working in the area of drug resistance in human tumor cells and, in particular, in the genetic mechanisms involved in the development of resistant human breast cancer cell lines. One, has increased levels of DHFR and amplified DHFR genes. We are using this gene amplified cell line to study the regulation of DHFR gene expression. We have also cloned the human DHFR gene and are planning to study the structural features which regulate this gene. Another human MTX resistant cell line is of particular interest, since it is defective in MTX polyglutamate formation. Studies in this cell line raise important questions regulating the mechanism of action of this drug and strongly suggest that MTX is in fact a pro-drug that needs to be metabolically converted to a more active form in order to exert its cytotoxicity. Both of these cell lines are being tested against a wide variety of antifolate analogues. Indeed the different mechanisms of resistance (DHFR overproduction and defective polyglutamate formation), result in marked differences in cross resistance and colateral sensitivity to other antifolate agents. We have also in collaboration studied the mechanisms of resistance in tumor specimens obtained from patients who have failed therapy with MTX and have shown that DHFR gene amplification does occur in vivo.